LysM Domain Research Articles

Cover Image:

Xu et al. (pages 235–248) demonstrate that OsCERK1 directly recognizes chitin molecules, which play a key role in mediating rice immunity against fungal pathogens and symbiotic relationships with AM fungi in rice. Structural information on chitin recognition by OsCERK1 can facilitate engineering of chitin receptors to enhance rice disease resistance. The cover image shows the crystal structure of OsCERK1‐ECD in complex with (NAG)6, in which the second LysM domain (LysM2) of OsCERK1‐ECD is the major binding site of the chitin oligomer.

Evaluation of the expression of induced genes in response to dehydration stress of Camelina (Camelina sativa) calli.

Plants are constantly exposed to various biological and non-biological stresses that endanger their lives. Drought stress is one of the abiotic stresses that have a great impact on the yield and life of plants and is one of the main causes of reduced crop yields. Reducing the effects of environmental stresses such as drought using methods such as irrigation, fertilizer application and appropriate planting methods is limited. Therefore, genetic modification of plants is an important effort to minimize the effect of environmental stresses. in this research, Twenty disinfected camelina seeds were cultured on the MS medium containing 3% sucrose, 0.8% agar and pH 5.8 under a laminar hood. After 14 days, the cotyledon explants (about 1 cm) were separated from the seedlings and placed on the callus induction medium. The MS callus induction medium containing 0.5 mg / l kinetin, 2 mg / l -2,4 D, 3% sucrose, 0.8% agar and pH 5.8. Samples were subcultured every two weeks to the same medium and calli were formed after 4 weeks. Then the calli were transferred to the medium containing a concentration of 30% PEG. To study gene expression, first callus samples were treated with liquid nitrogen and to study the effect of drought stress on gene expression, this sample was sent to Zagros Bioidea Company located in the Razi University Incubator. Gene expression was performed through microarray technology. The results showed that seven different genes whose expression increased by almost six times the control value can be mentioned, including Cold-acclimation protein (CAP160), NAC10, Abscisic acid (ABA), ABF4, CRK3, lysM domain receptor-like kinases (LYKs) and Basic/helix-loop-helix(bHLH130-like). Drought tolerance is not a genetically simple trait, but a quantitative and complex trait with various aspects that require the use of molecular methods to investigate the relevant mechanisms. This study aimed to investigate the expression of different genes of callus tissues of the Camelina plant under stress and non-stress conditions by microarray method.

Exploring the role of plant lysin motif receptor-like kinases in regulating plant-microbe interactions in the bioenergy crop Populus

For plants, distinguishing between mutualistic and pathogenic microbes is a matter of survival. All microbes contain microbe-associated molecular patterns (MAMPs) that are perceived by plant pattern recognition receptors (PRRs). Lysin motif receptor-like kinases (LysM-RLKs) are PRRs attuned for binding and triggering a response to specific MAMPs, including chitin oligomers (COs) in fungi, lipo-chitooligosaccharides (LCOs), which are produced by mycorrhizal fungi and nitrogen-fixing rhizobial bacteria, and peptidoglycan in bacteria. The identification and characterization of LysM-RLKs in candidate bioenergy crops including Populus are limited compared to other model plant species, thus inhibiting our ability to both understand and engineer microbe-mediated gains in plant productivity. As such, we performed a sequence analysis of LysM-RLKs in the Populus genome and predicted their function based on phylogenetic analysis with known LysM-RLKs. Then, using predictive models, molecular dynamics simulations, and comparative structural analysis with previously characterized CO and LCO plant receptors, we identified probable ligand-binding sites in Populus LysM-RLKs. Using several machine learning models, we predicted remarkably consistent binding affinity rankings of Populus proteins to CO. In addition, we used a modified Random Walk with Restart network-topology based approach to identify a subset of Populus LysM-RLKs that are functionally related and propose a corresponding signal transduction cascade. Our findings provide the first look into the role of LysM-RLKs in Populus-microbe interactions and establish a crucial jumping-off point for future research efforts to understand specificity and redundancy in microbial perception mechanisms.

Chitin-induced systemic disease resistance in rice requires both OsCERK1 and OsCEBiP and is mediated via perturbation of cell-wall biogenesis in leaves.

Chitin is a well-known elicitor of disease resistance and its recognition by plants is crucial to perceive fungal infections. Chitin can induce both a local immune response and a systemic disease resistance when provided as a supplement in soils. Unlike local immune responses, it is poorly explored how chitin-induced systemic disease resistance is developed. In this study, we report the systemic induction of disease resistance against the fungal pathogen Bipolaris oryzae by chitin supplementation of soils in rice. The transcriptome analysis uncovered genes related to cell-wall biogenesis, cytokinin signaling, regulation of phosphorylation, and defence priming in the development of chitin-induced systemic response. Alterations of cell-wall composition were observed in leaves of rice plants grown in chitin-supplemented soils, and the disease resistance against B. oryzae was increased in rice leaves treated with a cellulose biosynthesis inhibitor. The disruption of genes for lysin motif (LysM)-containing chitin receptors, OsCERK1 (Chitin elicitor receptor kinase 1) and OsCEBiP (Chitin elicitor-binding protein), compromised chitin-induced systemic disease resistance against B. oryzae and differential expression of chitin-induced genes found in wild-type rice plants. These findings suggest that chitin-induced systemic disease resistance in rice is caused by a perturbation of cell-wall biogenesis in leaves through long-distance signalling after local recognition of chitins by OsCERK1 and OsCEBiP.

Functions of lysin motif (LysM)-containing protein in antibacterial responses of sea cucumbers, Apostichopus japonicus

The lysin motif (LysM)-containing protein is one of widespread pattern-recognition receptors in prokaryotes and eukaryotes. Numerous LysM-containing gene sequences are present in gene databases; however, few have been well characterized, especially in echinoderms. In this study, the full-length cDNA of a novel LysM-containing gene was obtained from the sea cucumber Apostichopus japonicus, named AjLysM-1, using polymerase chain reaction (PCR) combined with rapid amplification of cDNA ends. We prepared and expressed recombinant AjLysM-1 protein (rAjLysM-1) and determined its pathogen-recognition ability by enzyme-linked immunosorbent and immunofluorescence assays. We also analyzed the tissue expression pattern and response to immune challenges of AjLysM-1 using quantitative real-time reverse transcription-PCR and in situ hybridization. The AjLysM-1 protein was predicted to be an intracellular non-secreted LysM-containing protein, highly homologous to the same protein in other marine echinoderms. AjLysM-1 transcripts were highest expressed in coelomocytes and were strikingly induced by challenge with representative bacterial and fungal polysaccharides. rAjLysM-1 showed weak binding to mannan, Pseudoalteromonas nigrifaciens, and Shewanella baltica, implying that AjLysM-1 might provide inadequate defense against Gram-negative bacteria and fungi. Notably, rAjLysM-1 also interacted with tyrosine protein kinase and filamin-B, indicating that it could be involved in focal adhesion in A. japonicus. These findings improve our understanding of the functions of LysM-containing proteins in marine echinoderms.

The sWT1+ - Transcript Confers Cytarabine Resistance Via Upregulation of HOXA3 and GATA2 Axis

The Wilms tumor gene (WT1) encodes for a zinc finger transcription factor which manifests as both tumor suppressive and oncogenic activities. Due to 3 translation start sites and alternative splicing at exon 5 and a lysine, threonine, and serine (KTS) motif, WT1 generates 12 major transcripts. WT1 expression is upregulated in ~90% of leukemia samples and is associated with chemoresistance and disease relapse. However, which WT1 transcripts are associated with chemoresistant has not been defined. We characterized that sWT1+/-, but not other transcripts decreased cell growth, downregulated CD71 expression, and conferred cytarabine resistance. Leukemia blast cells with low CD71 expression are relatively resistant to cytarabine, suggesting that CD71 could be used as a biomarker for cytarabine treatment. Subsequent RNAseq analysis revealed two transcription factors HOXA3 and GATA2 to be uniquely upregulated in sWT1+/-, but not in other transcripts. Consistent with the sWT1 +/- phenotype, HOXA3 or GATA2 overexpression decreased cell growth, reduced CD71 expression, and conferred cytarabine resistance. Real time-PCR analysis showed that overexpression of WT1 and GATA2 upregulates each other and HOXA3 expression; whereas overexpression of HOXA3 upregulates GATA2 expression. Furthermore, relapsed leukemia samples express a relatively higher level of HOXA3 and GATA2 compared to de novo AML samples in the BeatAML cohort. GATA2 and WT1 are also coexpressed in AML samples (TCGA AML and BeatAML). Interestingly, WT1 and GATA2 mutations are mutual exclusive in primary AML and MDS patient samples (TCGA AML, BeatAML, and MDS (MSKCC+ IWG, IPSSM, NEJM)). These data indicates sWT1+-, HOXA3, and GATA2 forms a transcript complex that regulate cytarabine sensitivity. As such, we have identified transcription factors and a biomarker for cytarabine sensitivity in AML.

Transcriptome analysis of maize pathogen Fusarium verticillioides revealed FvLcp1, a secreted protein with type-D fungal LysM and chitin-binding domains, that plays important roles in pathogenesis and mycotoxin production

Fusarium verticillioides is a key maize pathogen and produces fumonisins, a group of mycotoxins detrimental to humans and animals. Unfortunately, our understanding on how this fungus interacts with maize to trigger mycotoxin biosynthesis is limited. We performed a systematic computational network-based analysis of large-scale F. verticillioides RNA-seq datasets to identify gene subnetwork modules associated with virulence and fumonisin regulation. F. verticillioides was inoculated on two different maize lines, moderately resistant line hybrid 33K44 and highly susceptible line maize inbred line B73, to generate time-course RNA-Seq data. Among the highly discriminative subnetwork modules, we identified a putative hub gene FvLCP1, which encodes a putative a type-D fungal LysM protein with a signal peptide, three LysM domains, and two chitin binding domains. FvLcp1 is a unique protein that harbors these domains amongst five representative Fusarium species. FvLcp1 is a secreted protein important for fumonisin production with the LysM domain playing a critical role. The chitin-binding domain was essential for in vitro chitin binding. Using Magnaporthe oryzae, we learned that FvLcp1 accumulates in appressoria, suggesting that FvLcp1 is involved in host recognition and infection. Full length FvLcp1 suppressed BAX-triggered plant cell death in Nicotiana benthamiana. This unique type-D LysM secreted protein with a chitin-binding domain in F. verticillioides was shown to be potentially involved in suppressing host cell death and promoting fumonisin biosynthesis while the pathogen colonizes maize kernels.

Genome-wide identification, evolution analysis of LysM gene family members and their expression analysis in response to biotic and abiotic stresses in banana (Musa L.)

LysM (Lysin motif), in response to pathogenic molecular stresses, is a crucial signal recognition gene. To understand the molecular characteristics of banana LysM gene family members, we used a series of bioinformatics methods. Based on the genomic databases of Musa acuminata, Musa balbisiana and Musa itinerans, a total of 53 genes and 55 proteins were identified, with 21 genes and 23 proteins in the M.acuminata, 16 genes and 16 proteins in each of M.balbisiana and M.itinerans, respectively. According to the conserved structural domains, LysM can be divided into five classes, namely LysM&MltD, LYK, LYP, LysMn, and LysMe. The LysM gene was relatively highly conserved in the evolution of the three genomes of banana, and some differences occurred. Expression analysis revealed that MaLysM4-5 was relatively highly expressed under high-temperature stress, low-temperature stress and pathogen infection; at the same time, about one-third of the members were down-regulated under low-temperature stress and high-temperature stress, while the expression of MaLysM10-1 and MaLysM4-5 were up-regulated. After the banana wilt fungus FocTR4 infected the banana roots, MaLysM1 was down-regulated and MaLysM11-1 was up-regulated. In conclusion, our study suggests that MaLysMs may be necessary in the response to high- and low-temperature stresses, as well as the banana wilt fungus infestation. Overall, this paper found that LysM genes may be involved in biotic and abiotic stresses in banana, and provided helpful information about LysM’s evolution, expression and properties, which will provide theoretical references for further studies on the functions of LysM genes and resistance breeding in the future.

Fungal dual-domain LysM effectors undergo chitin-induced intermolecular, and not intramolecular, dimerization.

Chitin is a homopolymer of β-(1,4)-linked N-acetyl-D-glucosamine (GlcNAc) and a major structural component of fungal cell walls. In plants, chitin acts as a microbe-associated molecular pattern (MAMP) that is recognized by lysin motif (LysM)-containing plant cell surface-localized pattern recognition receptors (PRRs) that activate a plethora of downstream immune responses. To deregulate chitin-induced plant immunity and successfully establish infection, many fungal pathogens secrete LysM domain-containing effector proteins during host colonization. The LysM effector Ecp6 from the tomato (Solanum lycopersicum) leaf mold fungus Cladosporium fulvum can outcompete plant PRRs for chitin binding because two of its three LysM domains cooperate to form a composite groove with ultra-high (pM) chitin-binding affinity. However, most functionally characterized LysM effectors contain only two LysMs, including Magnaporthe oryzae MoSlp1, Verticillium dahliae Vd2LysM, and Colletotrichum higginsianum ChElp1 and ChElp2. Here, we performed modeling, structural, and functional analyses to investigate whether such dual-domain LysM effectors can also form ultra-high chitin-binding affinity grooves through intramolecular LysM dimerization. However, our study suggests that intramolecular LysM dimerization does not occur. Rather, our data support the occurrence of intermolecular LysM dimerization for these effectors, associated with a substantially lower chitin binding affinity than monitored for Ecp6. Interestingly, the intermolecular LysM dimerization allows for the formation of polymeric complexes in the presence of chitin. Possibly, such polymers may precipitate at infection sites to eliminate chitin oligomers, and thus suppress the activation of chitin-induced plant immunity.

Potential Biological Control of Endophytic Streptomyces sp. 5-4 Against Fusarium Wilt of Banana Caused by Fusarium oxysporum f. sp. cubense Tropical Race 4.

Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is one of the most disastrous fungal diseases. Biological control is a promising strategy for controlling Fusarium wilt of banana. To explore endophytic actinomycetes as biocontrol resources against Foc TR4, antagonistic strains were isolated from different tissues of medicinal plants. Here, a total of 144 actinomycetes were isolated and belonged to Nonomuraea, Kitasatospora, and Streptomyces. Forty-three isolates exhibited antifungal activities against Foc TR4. The strain labeled with 5-4 isolated from roots of Piper austrosinense had a broad-spectrum antifungal activity by the production of chitinase and β-1,3-glucanase and was identified as Streptomyces hygroscopicus subsp. hygroscopicus 5-4. Furthermore, disease index of banana wilt was significantly reduced by application of strain 5-4 in comparison with application of Foc TR4 alone. Exogenous application of strain 5-4 increased the expression levels of defense genes such as (PAL), peroxidase (POD), pathogenesis-related protein 1 (PR-1), hydrolytic enzymes (β-1,3-glucanase), lysin motif receptor kinase 1 (LYK-1), and mitogen-activated protein kinase 1 (MPK-1). The antifungal mechanism assay demonstrated that extracts of strain 5-4 inhibited spore gemination and hyphal growth of Foc TR4, and caused abnormally swollen, deformity, and rupture of Foc TR4 hypha. Thus, S. hygroscopicus subsp. hygroscopicus 5-4 could be used as a potential biological agent for controlling Fusarium wilt of banana.

Current Understanding of Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) Signaling in T-Cell Biology and Disease Therapy.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an immune checkpoint molecule that is mainly expressed on activated T cells and regulatory T (Treg) cells that inhibits T-cell activation and regulates immune homeostasis. Due to the crucial functions of CTLA-4 in T-cell biology, CTLA-4-targeted immunotherapies have been developed for autoimmune disease as well as cancers. CTLA-4 is known to compete with CD28 to interact with B7, but some studies have revealed that its downstream signaling is independent of its ligand interaction. As a signaling domain of CTLA-4, the tyrosine motif plays a role in inhibiting T-cell activation. Recently, the lysine motif has been shown to be required for the function of Treg cells, emphasizing the importance of CTLA-4 signaling. In this review, we summarize the current understanding of CTLA-4 biology and molecular signaling events and discuss strategies to target CTLA-4 signaling for immune modulation and disease therapy.

Promoting Resuscitation of the VBNC Bacteria and Enrichment of Naphthalene-degrading Bacteria from Oil-contaminated Soils with the Rpf4 from Rhodococcus erythropolis KB1

Nowadays, more than 90% of bacteria in nature are in a viable but not culturable (VBNC) state, and they cannot be separated by traditional plate culture methods. However, resuscitation-promoting factors (Rpf) participate in recovery of dormant cells and promoting bacterial growth. The Rpf4 of Rhodococcus erythropolis KB1 is a Rpf like protein of Micrococcus luteus and contained the Rpf domain and LysM domain. In this study, an expression vector for the rpf4 gene was constructed and expressed in Escherichia coli. The recombinant protein Rpf4 was purified by Ni2+-affinity chromatography. The recombinant protein showed a muralytic activity of 549 ± 28.77 U/mg. The purified Rpf4 could increase R. erythropolis KB1 cell proliferation and improve the resuscitation of R. erythropolis cells that were VBNC. The Rpf4 altered the diversity of naphthalene-degrading bacteria enriched from oil-contaminated soils. Based on the high-throughput sequencing, we found that Proteobacteria and Actinobacteria phyla increased from 76.10% and 0.029% to 98.23% and 0.401% after the Rpf4 treatment, while the Bacteroidetes phylum decreased from 22.70% to 0.76%. The genera Enterobacter, Citrobacter, and Acinetobacter increased from 0.0054%, 0.001%, and 0.745% to 19.02%, 6.34% and 2.49%. Some new genera such as Bifidobacterium, Limnobacter, Alloprevotella, Hydrogenophaga, and Nevskia were founded in the treatment group. Meanwhile, 23 naphthalene-degrading bacteria were isolated by adding the Rpf4 protein to the oil-contaminated soil samples. Study on the naphthalene-degrading bacteria will provide a basis for the microbial degradation of naphthalene and other polycyclic aromatic hydrocarbons and discover the wider application value of Rpf.

OsCERK2/OsRLK10, a homolog of OsCERK1, has a potential role for chitin-triggered immunity and arbuscular mycorrhizal symbiosis in rice.

In rice, the lysin motif (LysM) receptor-like kinase OsCERK1, originally identified as the essential molecule for chitin-triggered immunity, plays a key role in arbuscular mycorrhizal (AM) symbiosis. As we previously reported, although AM colonization was largely repressed at 2 weeks after inoculation (WAI), arbuscules were observed at 5 WAI in oscerk1 mutant. Conversely, most mutant plants that defect the common symbiosis signaling pathway exhibited no arbuscule formation. Concerning the reason for this characteristic phenotype of oscerk1, we speculated that OsRLK10, which is a putative paralog of OsCERK1, may have a redundant function in AM symbiosis. The protein sequences of these two genes are highly conserved and it is estimated that the gene duplication occurred 150 million years ago. Here we demonstrated that OsCERK2/OsRLK10 induced AM colonization and chitin-triggered reactive oxygen species production in oscerk1 knockout mutant as similar to OsCERK1. The oscerk2 mutant showed a slight but significant reduction of AM colonization at 5 WAI, indicating the contribution of OsCERK2 for AM symbiosis. However, the oscerk2;oscerk1 double-knockout mutant produced arbuscules at 5 WAI as similar to the oscerk1 mutant, indicating that the redundancy of OsCERK1 and OsCERK2 did not explain the mycorrhizal colonization in oscerk1 at 5 WAI. These results indicated that OsCERK2 has a potential to regulate both chitin-triggered immunity and AM symbiosis and at least partially contributes to AM symbiosis in rice though the contribution of OsCERK2 appears to be weaker than that of OsCERK1.

A LytM-Domain Factor, ActS, Functions in Two Distinctive Peptidoglycan Hydrolytic Pathways in E. coli.

Bacterial cell wall contains peptidoglycan (PG) to protect the cells from turgor and environmental stress. PG consists of polymeric glycans cross-linked with each other by short peptide chains and forms an elastic mesh-like sacculus around the cytoplasmic membrane. Bacteria encode a plethora of PG hydrolytic enzymes of diverse specificity playing crucial roles in growth, division, or turnover of PG. In Escherichia coli, the cross-link-specific endopeptidases, MepS, -M, and -H, facilitate the enlargement of PG sacculus during cell elongation, whereas LytM-domain factors, EnvC and NlpD activate the division-specific amidases, AmiA, -B, and -C to facilitate the cell separation. In a screen to isolate additional factors involved in PG enlargement, we identified actS (encoding a LytM paralog, formerly ygeR) as its overexpression compensated the loss of elongation-specific endopeptidase, MepS. The overexpression of ActS resulted in the generation of partly denuded glycan strands in PG sacculi, indicating that ActS is either an amidase or an activator of amidase(s). The detailed genetic and biochemical analyses established that ActS is not a PG hydrolase, but an activator of the division-specific amidase, AmiC. However, interestingly, the suppression of the mepS growth defects by actS is not mediated through AmiC. The domain-deletion experiments confirmed the requirement of the N-terminal LysM domain of ActS for the activation of AmiC, but not for the alleviation of growth defects in mepS mutants, indicating that ActS performs two distinctive PG metabolic functions. Altogether our results suggest that in addition to activating the division-specific amidase, AmiC, ActS modulates yet another pathway that remains to be identified.

β-Glucan and its nanocomposites in sustainable agriculture and environment: an overviewof mechanisms and applications.

β-Glucan is an eco-friendly, biodegradable, and economical biopolymer with important roles for acquiring adaptations to mitigate climate change in crop plants. β-Glucan plays a crucial role in the activation of functional plant innate immune system by triggering the downward signaling cascade/s, resulting in the accumulation of different pathogenesis-related proteins (PR-proteins), reactive oxygen species (ROS), antioxidant defense enzymes, Ca2+-influx as well as activation of mitogen-activated protein kinase (MAPK) pathway. Recent experimental studies have shown that β-glucan recognition is mediated by co-receptor LysMPRR (lysin motif pattern recognition receptor)-CERK1 (chitin elicitor receptor kinase 1), LYK4, and LYK5 (LysM-containing receptor-like kinase), as well as different receptor systems in plants that could be plant species-specific and/or age and/or tissue-dependent. Transgenic overexpression of β-glucanase, chitinase, and/or in combination with other PR-proteins like cationic peroxidase, AP24,thaumatin-likeprotein 1 (TLP-1) has also been achieved for improving plant disease resistance in crop plants, but the transgenic methods have some ethical and environmental concerns. In this regard, elicitation of plant immunity using biopolymer like β-glucan and chitosan offers an economical, safe, and publicly acceptable method. The β-glucan and chitosan nanocomposites have proven to be useful for the activation of plant defense pathways and to enhance plant response/systemic acquired resistance (SAR) against broad types of plant pathogens and mitigating multiple stresses under the changing climate conditions.

An integrated approach reveals how lipo-chitooligosaccharides interact with the lysin motif receptor-like kinase MtLYR3.

N‐acetylglucosamine containing compounds acting as pathogenic or symbiotic signals are perceived by plant‐specific Lysin Motif Receptor‐Like Kinases (LysM‐RLKs). The molecular mechanisms of this perception are not fully understood, notably those of lipo‐chitooligosaccharides (LCOs) produced during root endosymbioses with nitrogen‐fixing bacteria or arbuscular mycorrhizal fungi. In Medicago truncatula, we previously identified the LysM‐RLK LYR3 (MtLYR3) as a specific LCO‐binding protein. We also showed that the absence of LCO binding to LYR3 of the non‐mycorrhizal Lupinus angustifolius, (LanLYR3), was related to LysM3, which differs from that of MtLYR3 by several amino acids and, particularly, by a critical tyrosine residue absent in LanLYR3. Here, we aimed to define the LCO binding site of MtLYR3 by using molecular modelling and simulation approaches, combined with site‐directed mutagenesis and LCO binding experiments. 3D models of MtLYR3 and LanLYR3 ectodomains were built, and homology modelling and molecular dynamics (MD) simulations were performed. Molecular docking and MD simulation on the LysM3 identified potential key residues for LCO binding. We highlighted by steered MD simulations that in addition to the critical tyrosine, two other residues were important for LCO binding in MtLYR3. Substitution of these residues in LanLYR3‐LysM3 by those of MtLYR3‐LysM3 allowed the recovery of high‐affinity LCO binding in experimental radioligand‐binding assays. An analysis of selective constraints revealed that the critical tyrosine has experienced positive selection pressure and is absent in some LYR3 proteins. These findings now pave the way to uncover the functional significance of this specific evolutionary pattern.

Structure, mechanism, and phylogeny of LysM-chitinase conjugates specifically found in fern plants

A unique GH18 chitinase containing two N-terminal lysin motifs (PrLysM1 and PrLysM2) was first found in fern, Pteris ryukyuensis (Onaga and Taira, Glycobiology, 18, 414–423, 2008). This type of LysM-chitinase conjugates is not usually found in plants but in fungi. Here, we produced a similar GH18 chitinase with one N-terminal LysM module (EaLysM) from the fern, Equisetum arvense (EaChiA, Inamine et al., Biosci. Biotechnol. Biochem., 79, 1296–1304, 2015), using an Escherichia coli expression system and characterized for its structure and mechanism of action. The crystal structure of EaLysM exhibited an almost identical fold (βααβ) to that of PrLysM2. From isothermal titration calorimetry and nuclear magnetic resonance, the binding mode and affinities of EaLysM for chitooligosaccharides (GlcNAc)n (3, 4, 5, and 6) were found to be comparable to those of PrLysM2. The LysM module in EaChiA is likely to bind (GlcNAc)n almost independently through CH-π stacking of a Tyr residue with the pyranose ring. The (GlcNAc)n-binding mode of LysMs in the LysM-chitinase conjugates from fern plants appears to differ from that of plant LysMs acting in chitin- or Nod-signal perception, in which multiple LysMs cooperatively act on (GlcNAc)n. Phylogenetic analysis suggested that LysM-GH18 conjugates of fern plants formed a monophyletic group and had been separated earlier than forming the clade of fungal chitinases with LysMs.

Multi-epitope based vaccine design against Staphylococcus epidermidis: A subtractive proteomics and immunoinformatics approach

Staphylococcus epidermidis has emerged as a major contributor of nosocomial infections across the world. With the increased rate of emerging resistant and previously undefined infectious diseases, there is a growing need to develop a novel vaccine possessing required immunogenic properties. The adopted reverse vaccinology approach identified “IMPNQILTI” of LysM domain protein, “YSYTYTIDA” of staphylococcal secretory antigen SsaA, and “YNYDANTGQ” neutral metalloproteinaseas potential peptides for vaccine design. The 9-mer epitope of target proteins is antigenic, virulent, surface-exposed, non-allergenic, and conserved across various strains of S. epidermidis. Protein-protein interactions study indicated the involvement of target proteins in major biological pathways for S. epidermidis pathogenesis. Protein-peptide docking was performed, and population coverage analysis showed significant interactions of T-cell epitopes with the HLA-binding molecules while covering 90.58% of the world's population. Further, a multi-epitope vaccine of 177 amino acids long was constructed. Docking with Toll-like receptor (TLR-2) molecule confirmed the effective interaction of the vaccine with the receptor. The vaccine efficiency in generating an effective immune response in the host was evaluated by immune simulation. Finally, in silico cloning confirmed that the constructed vaccine can be efficiently expressed in E. coli. However, the designed vaccine needs experimental validation to determine the effectiveness and immunogenicity profile, which will ensure an active immunity against S. epidermidis.

Knockdowns of red Malphigian tubules reveal pigmentation roles in the milkweed bug.

Classical Drosophila eye color mutations have unearthed a toolkit of genes that have permitted candidate gene studies of the outstanding diversity of coloration patterns in other insects. The gene underlying the eye color phenotypes of the red Malphigian tubules (red) fly mutant was mapped to a LysM domain gene of unknown molecular function. Here, we used RNAi to test the role of a red ortholog in the pigmentation of the milkweed bug Oncopeltus fasciatus, and contrast its effect with the ommochrome biosynthetic pathway gene vermilion (ver). Pigmentation was reduced in the cuticle of embryonic legs and first instar abdomens following parental RNAi against red, but not against ver, likely reflecting an effect on pterin biogenesis. Nymphal RNAi of red and ver both resulted in adult eye depigmentation, consistent with an effect on ommochrome content. These results suggest red loss-of-function impacts biochemically distinct types of pigments, and we discuss its putative role in the biogenesis of lysosome-related organelles such as ommochromasomes and pterinosomes.

A marine chitinase from Bacillus aryabhattai with antifungal activity and broad specificity toward crystalline chitin degradation

Chitinases convert chitin into chitin oligomers and are also known antifungal agents. Chitin oligomers have numerous industrial applications. However, chitin’s crystalline nature requires pretreatment before breakdown into oligomers. In the study, a novel marine bacterium Bacillus aryabhattai is isolated from the Arabian Sea. Bacterial growth in different crystalline chitin substrates like chitin powder, chitin flakes, and colloidal chitin confirmed the chitinase presence in bacterium could act upon insoluble crystalline chitin with the fractional release of oligomers. The domain architecture analysis of the chitinase confirmed the presence of two N-terminal LysM domains which help enzyme action on crystalline chitin. Statistical optimization of media and Process parameters revealed glycerol, yeast extract, magnesium chloride, and manganese sulfate as significant media components along with colloidal chitin. The optimum process parameters such as pH 7, temperature 40 °C, inoculum size 12.5% (v/v), and inoculum age 20 hours enhanced the specific enzyme activity to ±146.2 U/mL, ±114.9 U/mL and ±175.4 U/mL against chitin powder, chitin flakes and colloidal chitin respectively, which is five to six times higher than basal level activity. The antifungal activity of chitinase against plant pathogenic fungi like Candida albicans and Fusarium oxysporum revealed a zone of inhibition with 14 mm diameter.